The epidermal growth factor receptor-2 (HER2, ErbB2) modulates its activity through a tyrosine kinase signaling pathway and is involved in the development of various types of cancers such as those of the lungs, breast, head and neck, etc (1, 2). Overexpression or amplification of the HER2 gene is known to occur in a high percentage of cancer cases (e.g., ~20% of breast cancer) and predicts a poor prognosis for the patient. Invasive methods such as biopsies in conjunction with immunohistochemistry and fluorescence in situ hybridization are often employed to assess the HER2 status of the primary and metastasized neoplastic tumors; however, because of sampling bias and tumor heterogeneity, results obtained with these procedures may not be reliable (1). Because of their small size and high chemical and thermal stability, there is much interest in the use of radiolabeled Affibodies (an Affibody molecule is a chain of 58 amino acids (~7 kDa) that contains a modified B domain of the staphylococcal protein A and can be obtained by chemical synthesis or produced in bacteria with the use of recombinant DNA technology (3)) for the targeted detection and treatment of malignant tumors as discussed in detail elsewhere (3-5). A biodistribution study using 111In-labeled anti-epidermal growth factor receptor (EGFR) Affibody (ZHER:1907; the radionuclide was chelated to the Affibody through a metal chelator) with single-photon emission computed tomography (SPECT) showed that the tracer was suitable for the molecular imaging of EGFR expression in xenograft tumors in mice (6). However, a very high accumulation of radioactivity from the labeled Affibody was found in the kidneys and suggested that the tracer should not be used to detect cancerous tumors in close proximity of these organs. Ekblad et al. showed that the position of a metal chelator on the Affibody affected the in vivo stability and biodistribution of the molecule (7).
It was reported that an anti-EGFR Affibody dimer ((ZHER2:342)2) bearing a hexa-histidine tag (H6) on the N-terminus (H6-(ZHER2:342)2) can be labeled with 99mTc-tricarbonyl ([99mTc(CO)3]+), and the radiolabeled Affibody ([99mTc(CO)3]+-H6-(ZHER2:342)2) can be used to visualize tumors expressing HER2 in mice (8). With this construct, a higher amount of radioactivity was observed to accumulate in the liver compared with the tumors, and the investigators concluded that, because cancers usually metastasize to the liver, [99mTc(CO)3]+-H6-(ZHER2:342)2 was suitable only for the imaging of extrahepatic tumors. Similar observations were also made with other anti-EGFR Affibodies (9). On the basis of a hypothesis that the uptake of a labeled compound by the liver can be reduced by removing the H6 tag from the N-terminus of the ZHER2:342 Affibody or by increasing the hydrophilicity of the tag, two new tracers were constructed by Tolmachev et al. (10). In one construct, the H6 tag was moved from the N-terminal of the Affibody to the C-terminal (ZHER2:342-H6); in the second construct, the tag located on the N-terminal of the Affibody was made more hydrophilic by substituting it with a glutamatic acid-histidine trimer ((HE)3) to generate (HE)3-ZHER2:342. These constructs were subsequently labeled with [99mTc(CO)3]+ to form [99mTc(CO)3]+-ZHER2:342-H6 and [99mTc(CO)3]+-(HE)3-ZHER2:342, respectively. The biodistribution patterns of these radiolabeled Affibodies were studied in normal and nude mice bearing LS174T cell xenograft tumors (the LS174T is a human colorectal cancer cell line that has a low expression of HER2) and compared it with that of the parent radiolabeled Affibody ([99mTc(CO)3]+-H6-ZHER2:342) described previously (11). Results from this study showed that the accumulation of radioactivity in the liver with the ZHER2:342-H6 and the (HE)3-ZHER2:342 constructs was almost 10-fold lower than that with H6-ZHER2:342, and there was a similar uptake of label in the tumors with all three conjugates. In addition, the tumor/liver radioactivity ratio with [99mTc(CO)3]+-(HE)3-ZHER2:342 was far superior to that observed with the other constructs.
On the basis of observations described above, it was postulated that the use of a (HE)3-tag in an anti-HER2 Affibody may be useful even if the C-terminus of the molecule is modified by the addition of a cysteine (C) residue for site-specific labeling of the peptide (12). To demonstrate this, three new Affibody constructs were prepared (H6-ZHER2:342-C, ZHER2:342-H6-C, and (HE)3-ZHER2:342-C) and labeled with 111In, 99mTc, and 125I, respectively. The biodistribution of the various radiolabeled Affibodies ([111In/125I/99mTc]-labeled (HE)3-ZHER2:342-C, [111In/125I/99mTc]-labeled H6-ZHER2:342-C, and [111In/125I/99mTc]-labeled ZHER2:342-H6-C) was then studied in normal mice.